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Bone 42 (2008) 914 920 www.elsevier.

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Continuous PGE2 leads to net bone loss while intermittent PGE2 leads to net bone gain in lumbar vertebral bodies of adult female rats
X.Y. Tian a , Q. Zhang a , R. Zhao a , R.B. Setterberg a , Q.Q. Zeng b , S.J. Iturria b,c , Y.F. Ma b , W.S.S. Jee a,
a

Division of Radiobiology, Department of Radiology, University of Utah School of Medicine, 729 Arapeen Dr., Suite 2338, Salt Lake City, UT, 84108-1218, USA b Department of Endocrinology, Musculoskeletal Research, Lilly Corporate Center, Indianapolis, IN, USA c Global Statistical Sciences Discovery Statistics, Lilly Corporate Center, Indianapolis, IN, USA Received 9 July 2007; revised 19 November 2007; accepted 12 December 2007 Available online 5 February 2008

Abstract The present study examined the effects of continuous and intermittent PGE2 administration on the cancellous and cortical bone of lumbar vertebral bodies (LVB) in female rats. Six-month-old SpragueDawley female rats were divided into 6 groups with 2 control groups and 1 or 3 mg PGE2/kg given either continuously or intermittently for 21 days. Histomorphometric analyses were performed on the cancellous and cortical bone of the fourth and fifth LVBs. Continuous PGE2 exposure led to bone catabolism while intermittent administration led to bone anabolism. Both routes of administration stimulated bone remodeling, but the continuous PGE2 stimulated more than the intermittent route to expose more basic multicellular units (BMUs) to the negative bone balance. The continuous PGE2 caused cancellous bone loss by stimulating bone resorption greater than formation (i.e., negative bone balance) and shortening the formation period. It caused more cortical bone loss than gain, the magnitude of the negative endocortical bone balance and increased intracortical porosity bone loss was greater than for periosteal bone gain. The anabolic effects of intermittent PGE2 resulted from cancellous bone gain by positive bone balance from stimulated bone formation and shortened resorption period; while cortical bone gain occurred from endocortical bone gain exceeding the decrease in periosteal bone and increased intracortical bone loss. Lastly, a scheme to take advantage of the marked PGE2 stimulation of lumbar periosteal apposition in strengthening bone by converting it to an anabolic agent was proposed. 2008 Elsevier Inc. All rights reserved.
Keywords: Continuous PGE2; Intermittent PGE2; Negative and positive; Endosteal bone balance; Periosteal expansion

Introduction Numerous in vivo studies have identified prostaglandin of the E series (PGE) as a potent anabolic agent that stimulates both modeling (i.e. formation drift on quiescent surface) and remodeling-dependent (i.e. positive basic multicellular unit [BMU] bone balance) bone gain when delivered intermittently by daily subcutaneous injections [18]. However, there is limited knowledge on the tissue and cellular level changes of continuous PGE administration. There are several reports of continuous infusion of PGE1 in human infants with congenital
Corresponding author. Fax: +1 801 581 7008. E-mail address: webster.jee@hsc.utah.edu (W.S.S. Jee). 8756-3282/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2007.12.228

heart disease in which PGE1 administration resulted in cortical hyperostosis of the long bones due to periosteal bone formation but lacking detailed histomorphometric analysis [911]. Only one pre-clinical study exists in which PGE2 was administered by subcutaneous implantation of a controlled release pellet in 7-week-old female rats [12]. In this model, there was cancellous bone loss but no other response. This route of administration induced inflammation that led these investigators to conclude that the pellet administration route does not provide a reliable method of systemic PGE2 administration. This lack of literature indicates a need for a pre-clinical study of the effect of continuous PGE2 administration on lumbar vertebral bodies. The current study was designed to compare the effects of continuous PGE2 infusion in 6-month-old female rats to the

X.Y. Tian et al. / Bone 42 (2008) 914920 Table 1 Static and dynamic histomorphometric changes in the cancellous bone of LVB4 + LVB5 Groups Baseline Control Continuous 1 mg Continuous 3 mg Intermittent 1 mg Intermittent 3 mg %B.Ar (%) 33.27 5.74 33.80 4.70 26.56 2.14a ( 21) 25.96 3.19a ( 23) 36.93 6.00bc (9) 37.50 3.91abc (11) Tb.Wi (m) 73.44 7.51 72.74 5.64 60.27 4.62a ( 17) 59.33 6.32a ( 18) 81.64 8.95abc (12) 82.93 9.24abc (14) Tb.N (#/mm) 4.53 0.69 4.65 0.59 4.42 0.43 ( 5) 4.39 0.43 ( 6) 4.54 0.70 ( 2) 4.57 0.70 ( 2) Tb.Sp (m) 151.66 33.56 145.62 29.33 167.68 19.09a (15) 170.54 21.13a (17) 143.04 31.45bc (2) 140.23 26.49bc (4) %Er.Pm (%) 1.14 0.39 1.10 0.39 2.30 0.72a (110) 2.86 0.75a (161) 1.00 0.25bc ( 9) 1.20 0.36bc (9) %L.Pm (%) 32.92 5.13 33.14 4.08 31.21 3.19 ( 6) 38.09 7.76ab (15) 45.12 8.37abc (36) 50.09 2.75abcd (51) MAR (m/d) 1.00 0.08 0.98 0.08 0.98 0.12 (0) 1.12 0.10ab (15) 1.15 0.11ab (17) 1.06 0.10ad (9)

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BFR/BS (m3/m2/d) 32.98 6.46 32.31 4.70 30.36 3.97 ( 6) 42.26 7.45ab (31) 51.61 10.16abc (60) 53.19 5.09abc (65)

Mean SD. % change from control in parentheses. Note. pb0.05 vs. acontrol, bcontinuous 1 mg, ccontinuous 3 mg, dintermittent 1 mg. LVB4: Fourth lumbar vertebral body longitudinal section. LVB5: Fifth lumbar vertebral body cross section. Continuous 1 mg and 3 mg: 1 and 3 mg PGE2/kg/d continuous infusion; intermittent 1 mg and 3 mg: 1 and 3 mg PGE2/kg/d daily sc injection. %B.Ar: percent trabecular bone area, Tb.Wi: trabecular width, Tb.N: trabecular number, Tb.Sp: trabecular separation, %Er.Pm: percent eroded perimeter, % L.Pm: percent mineralized perimeter, MAR: mineral apposition rate, BFR/BS: bone formation rate per unit of bone surface.

well-established effects of intermittent administration. Histomorphometric analysis was performed on the lumbar vertebrae to profile tissue and cellular changes of bone gain or loss. In addition, we propose a scheme to take advantage of the fact that continuous PGE2 stimulates periosteal apposition to increase bone strength to convert continuous administration PGE2 to an anabolic agent.
Materials and methods Experimental protocol
Forty-eight, 6-month-old, female SpragueDawley rats (Harlan Sprague Dawley Inc., Indianapolis, IN), weighing approximately 220 g, were maintained on a 12-h light/12-h dark cycle at 22 C with ad libitum access to food (TD 5001 with 0.95% calcium and 0.67% phosphorus, vitamin D3 4500 IU/kg; Teklad, Madison, WI) and water. Eight rats were sacrificed as the baseline + 9 days and aging controls, while the remaining rats were randomly divided into 4 groups with eight rats in each group. Prostaglandin E2 (Upjohn Company, Kalamazoo, MI) at 1 or 3 mg/kg/d was given to the rats by continuous infusion via InfuDisk pump (Med-E-Cell, San Diego, CA) or daily subcutaneous (sc) injection on the back for 21 days. The Infu-Disks were connected to the jugular vein, Table 2 Bone remodeling and bone balance changes in cancellous bone of LVB4 + LVB5 Groups BFR/BV (%/yr) W.Wi (m) FP (d) RP (d)

and placed on the back with a special jacket and changed every 7 days. Prostaglandin E2 was first dissolved in ethanol then further diluted into the final injection solution (10% ethanol with 1 ml/kg injection volume). All rats received sc injections with Calcein 5 mg/kg (Sigma, St Louis, MO) on days 10, 9, 3 and 2 before sacrifice. The Institutional Animal Care and Use Committee of Lilly Research Laboratories approved the animal protocol to ensure compliance with the NIH animal care guidelines. At necropsy, the fourth and fifth lumbar vertebrae were stained with Villanueva osteochrome bone stain (Arizona Histology & Histomorphometry Center, Phoenix, AZ, USA), and embedded in methyl methacrylate. Sawed and ground 20-m longitudinal lumbar sections (LVB4), and 30-m cross lumbar sections (LVB5) were prepared for analyses. Histomorphometric measurements were acquired by using an Image Analysis System (Osteomeasure, Inc., Atlanta, GA). Cancellous bone analysis was performed in the LVB4 longitudinal sections, which included the spongiosa between 0.1 mm from the cranial and caudal growth plates; while in the LVB5 cross sections analysis was limited to the spongiosa of the body and the ventral cortices [7,1315]. The data gathered from the LVB4 and LVB5 sections were very similar so the data were combined for analyses. All data were presented as a group mean standard deviation (SD). The statistical analyses were performed using the Ultimate Integrated Data Analysis and Presentation System (StatView 5.0.1, SAS Institute Inc. Cary, NC, USA). Across group comparisons were made with a parametric analysis of variance (ANOVA); p b 0.05 was considered significant. The one-sample KolmogorovSmirnov test for normality was applied to each

Rm.P (d)

Act.F (cycle/yr)

%L.Pm %Er.Pm

BFR/BS %Er.Pm 33.17 15.14 34.29 17.53 14.73 5.84a ( 57) 15.51 4.00a ( 55) 54.22 14.85abc (58) 48.99 17.47abc (43)

Baseline Control Continuous 1 mg Continuous 3 mg Intermittent 1 mg Intermittent 3 mg

276.86 63.32 272.26 48.05 308.77 48.42 (13) 433.17 53.84ab (59) 385.47 66.53abc (42) 397.36 76.08ab (46)

12.43 0.89 12.88 0.95 12.58 0.91 ( 2) 12.76 1.22 ( 1) 13.66 1.29 (6) 13.88 1.23abc (8)

12.53 1.50 13.31 1.71 13.13 2.39 (1) 11.51 1.83a (14) 12.05 1.90 (9) 13.20 2.03c (1)

2.52 1.60 2.63 1.58 3.34 1.69 (27) 2.64 0.74 (1) 1.12 0.36abc (57) 1.69 0.67abcd (36)

15.06 2.93 15.93 3.12 16.47 3.96 (3) 14.15 2.29 ( 11) 13.18 2.20ab ( 17) 14.89 2.62 ( 7)

2.00 0.64 1.90 0.67 2.82 0.79a (49) 4.00 0.45ab (111) 3.39 0.85ac (79) 2.72 0.44acd (43)

32.70 13.65 34.60 16.09 15.00 5.52a ( 57) 13.93 3.83a ( 60) 47.09 11.50abc (36) 45.30 13.26abc (31)

Mean SD. % change from control in parentheses. Note. p b 0.05 vs. acontrol, bcontinuous 1 mg, ccontinuous 3 mg, dintermittent 1 mg. LVB4: Fourth lumbar vertebral body longitudinal section. LVB5: Fifth lumbar vertebral body cross section. Continuous 1 mg and 3 mg: 1 and 3 mg PGE2/kg/d continuous infusion; intermittent 1 mg and 3 mg: 1 and 3 mg PGE2/kg/d daily sc injection. BFR/BV: bone formation rate per unit of bone volume; W.Wi: wall width. FP: formation period, RP: resorption period, Rm.P: remodeling period, Act.F: activation frequency. %L.Pm/%Er.Pm: ratio of percent mineralized perimeter to percent eroded perimeter; BFR/BS/%Er.Pm: ratio of bone surface bone formation rate to percent eroded perimeter.

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Fig. 1. Von Kossa stained cross sections of fifth lumbar vertebral body from (A) control, (B) continuous treatment of 3 mg PGE2/kg/d and (C) intermittent treatment of 3 mg PGE2/kg/d. Continuous treatment (B) reduced trabecular bone mass, trabecular thickness and cortical thickness; intermittent treatment (C) increased trabecular bone mass and trabecular thickness. combination of response variables and measurement sites. In no instance was there a statistically significant departure from a normal distribution (all p values N 0.05) [16].

Results Significant changes ( p b 0.05) in lumbar vertebral cancellous bone Continuous 1 mg PGE2/kg for 21 days decreased cancellous bone (%B.Ar 21%) and architecture (Tb.Wi 17%, Tb.Sp + 15%) compared to aging control (Table 1). Its histomorphometric profile between 11 and 21 days exhibited significantly increased bone resorption (%Er.Pm + 110%; Table 1), bone remodeling (Act.F + 49%) resulting in a negative cancellous bone balance (%L.Pm/%Er.Pm and BFR/BS/%Er.Pm of 57%) (Table 2). Continuous 3 mg PGE2/kg significantly reduced cancellous bone (%B.Ar 23%) and architecture (Tb.Wi 18% and Tb.Sp +17%) (Table 1; Figs. 1B and 2B). The histomorphometric profile consisted of significantly elevated bone resorption (%Er.Pm +161%) greater than formation (%L.Pm +15%, MAR +15% and BFR/BS +31%) (Table 1) that increased bone remodeling (Act.F +111%) and turnover (BFR/BV +59%) accompanied by a decreased formation period (FP -14%), which contributed to the

negative bone balance (%L.Pm/%Er.Pm 60% and BFR/BS/%Er. Pm 55%) (Table 2). In contrast, intermittent 1 mg PGE2/kg significantly increased trabecular thickness only (Tb.Wi +12%; Table 1). Its histomorphometric profile included stimulated bone formation (%L.Pm +36%, MAR +17% and BFR/BS +60%) (Table 1) that increased bone remodeling (Act.F +79%), turnover (BFR/BV +42%), and decreased resorption and remodeling periods (RP 57% and Rm. P 17%), which contributed to a positive cancellous bone balance (%L.Pm/%Er.Pm +36% and BFR/BS/%Er.Pm +43%) (Table 2). Intermittent 3 mg PGE2/kg increased cancellous bone mass (%B.Ar + 11%), trabecular thickness (Tb.Wi +14%) (Table 1), and wall width (W.Wi + 8%; Table 2). Its histomorphometric profile was made up of stimulated bone formation (%L.Pm +51%, MAR +9% and BFR/BS +65%; Table 1; Figs. 1C and 2C) that activated bone remodeling (Act.F +43%) and turnover (BFR/ BV +46%), while decreasing resorption period (RP 36%), which contributed toward a positive cancellous bone balance (%L.Pm/%Er.Pm +31% and BFR/%Er.Pm +43%) (Table 2). Significant changes (p b 0.05) in lumbar vertebral cortical bone Continuous 1 mg PGE2/kg decreased cortical bone mass (%Ct.B.Ar 17%, %Ma.Ar + 10%, Ct.Th 18% and Ic-%

Fig. 2. Villanueva bone stained cross sections of fifth lumbar vertebral body under fluorescent microscope from (A) control, (B) continuous 3 mg PGE2/kg/d and (C) intermittent 3 mg PGE2/kg/d showing morphology and fluorochrome labeling. Continuous treatment (B) resulted in fewer and thinner trabeculae with increased eroded perimeter and labeling and thinner cortical thickness but stimulated periosteal new bone formation (arrow) and increased intracortical porosity (asterisks). Intermittent PGE2 treatment (C) caused increased and thicker trabeculation, abundant double labeled surfaces with formation greater than resorption, but with decreased periosteal bone formation.

X.Y. Tian et al. / Bone 42 (2008) 914920 Table 3 Static and dynamic histomorphometric and periosteal bone formation changes in cortical bone of LVB5 Groups Baseline Control Continuous 1 mg Continuous 3 mg Intermittent 1 mg Intermittent 3 mg T.Ar (mm2) 3.78 0.37 3.45 0.14 3.64 0.39 (6) 3.44 0.32 (0) 3.62 0.76 (5) 3.65 0.44 (6) %Ma.Ar (%) 60.36 4.58 60.82 1.30 67.19 4.84a (10) 68.22 3.75a (12) 58.43 2.74bc ( 4) 59.44 0.86bc ( 2) %Ct.B.Ar (%) 39.32 4.52 38.86 1.26 32.44 4.80a ( 17) 31.25 3.65a ( 20) 41.10 2.75bc (6) 39.73 1.19bc (2) Ct.Th (m) 315.81 35.65 305.95 7.21 252.20 26.14a ( 18) 235.62 35.99a ( 23) 285.67 42.42c ( 7) 295.73 21.92bc ( 3) Ic-%Po.Ar (%) 0.81 0.22 0.84 0.19 1.13 0.16a (34) 1.66 0.56ab (97) 1.14 0.18a (36) 1.37 0.34a (63) Ps-%L.Pm (%) 59.48 8.63 54.21 11.61 69.33 13.07 (28) 77.38 10.08a (43) 34.75 6.04abc ( 36) 42.98 2.62abcd ( 21) Ps-MAR (m/d) 0.99 0.01 0.90 0.12 1.39 0.08a (54) 1.47 0.25a (63) 0.43 0.25abc (52) 0.38 0.21abc (58)

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Ps-BFR (m/d 100) 58.92 8.94 49.47 14.31 96.49 21.05a (95) 114.93 30.02a (132) 15.95 10.13abc ( 68) 16.55 9.59abc ( 67)

Mean SD. % change from control in parentheses. Note. p b 0.05 vs. acontrol, bcontinuous 1 mg, ccontinuous 3 mg, dintermittent 1 mg. LVB5: Fifth lumbar vertebral body cross section. Continuous 1 mg and 3 mg: 1 and 3 mg PGE2/kg/d continuous infusion; intermittent 1 mg and 3 mg: 1 and 3 mg PGE2/kg/d daily sc injection. T.Ar: total tissue area, %Ma.Ar: percent marrow area, Ct.Th: cortical thickness. Ic: Intracortical, Ic-%Po.Ar: percent intracortical porosity area, Ps-: Periosteal surface. Ps-%L.Pm: percent periosteal mineralized perimeter, Ps-MAR: periosteal mineral apposition rate, Ps-BFR: bone formation rate per unit of periosteal surface.

Po.Ar + 34%; Table 3). The endocortical bone loss from increased endocortical bone resorption and negative endocortical bone balance (Ec-%Er.Pm +111%, Ec-%L.Pm/%Er. Pm 52%, and Ec-BFR/%Er.Pm 53%; Table 4) exceeded the stimulated periosteal bone formation (Ps-MAR +54% and Ps-BFR + 95%; Table 3). Continuous 3 mg PGE2/kg decreased cortical bone mass (% Ct.B.Ar 20%, %Ma.Ar +12%, Ct.Th 23%, and Ic-%Po.Ar + 97%; Table 3; Fig. 2B). Endocortical bone loss occurred by a stimulated bone resorption (Ec-%Er.Pm + 321%) and a negative endocortical bone balance (Ec-%L.Pm/%Er.Pm 75% and EcBFR/%Er.Pm 76%; Table 4) that exceeded the stimulated periosteal bone formation (Ps-%L.Pm + 43%, MAR + 63% and BFR +132%; Table 3). Intermittent 1 mg PGE2/kg lacked effect on mass but increased intracortical porosity (Ic-%Po.Ar +36%; Table 3). The endocortical bone gain from stimulated endocortical bone formation (Ec-%L.Pm + 123%, MAR + 37% and BFR + 196%) and positive endocortical bone balance (Ec-%L.Pm/%Er.Pm +125% and Ec-BFR/%Er.Pm + 200%) (Table 4) was offset by increased intracortical porosity (Ic-%Po.Ar + 36%) and
Table 4 Endocortical surface histomorphometric changes in cortical bone of LVB5 Groups Baseline Control Continuous 1 mg Continuous 3 mg Intermittent 1 mg Intermittent 3 mg Ec-%Er.Pm (%) 3.17 0.38 3.09 0.28 6.50 2.15a (111) 12.98 5.20ab (321) 3.06 0.35bc ( 1) 3.41 0.35bc (11) Ec-%L.Pm (mm) 24.29 4.83 22.49 7.25 20.85 3.78 ( 7) 17.20 4.36 ( 24) 50.07 4.43abc (123) 50.69 6.23abc (125)

decreased periosteal bone formation (Ps-%L.Pm 36%, MAR 52% and BFR 68%; Table 3). Intermittent 3 mg PGE2/kg also lacked effect on cortical bone but did increase intracortical porosity (Ic-%Po.Ar +63%; Table 3; Fig. 2C). Again, the endocortical bone gain from stimulated bone formation (Ec-%L.Pm + 125%, MAR +36% and BFR + 200%; Table 4) was offset by the increased intracortical porosity (Ic-%Po.Ar +63%) and by the decreased periosteal bone formation (Ps-%L.Pm 25%, MAR 58% and BFR 67%; Table 3). Discussion The net response of cancellous bone to continuous administration of PGE2 was catabolic. In as little as 21 days, it induced cancellous bone loss by stimulated bone turnover and shortened formation and remodeling periods combined with negative cancellous bone balance to yield an imbalance of resorption and formation in favor of resorption. In cortical bone, the bone loss was due to an imbalance of bone loss over gain. The loss from negative endocortical bone balance and increased intracortical

Ec-MAR (m/d) 0.79 0.14 0.78 0.12 0.79 0.05 (2) 0.82 0.17 (6) 1.06 0.05abc (37) 1.06 0.11abc (36)

Ec-BFR (m/d 100) 19.17 5.69 18.02 8.20 16.57 3.30 ( 8) 13.81 2.96 ( 23) 53.29 5.31abc (196) 54.13 11.25abc (200)

Ec-%L.Pm/%Er.Pm 7.87 2.57 7.33 2.39 3.53 1.36a ( 52) 1.82 1.68a ( 75) 16.52 1.95abc (125) 15.05 2.78abc (105)

Ec-BFR/%Er.Pm 6.32 2.91 5.86 2.67 2.78 1.00a ( 53) 1.40 1.06ab ( 76) 17.59 2.20abc (200) 16.15 4.35abc (176)

Mean SD. % change from control in parentheses. Note. p b 0.05 vs. acontrol, bcontinuous 1 mg, ccontinuous 3 mg, dintermittent 1 mg. LVB5: Fifth lumbar vertebral body cross section. Continuous 1 mg and 3 mg: 1 and 3 mg PGE2/kg/d continuous infusion; intermittent 1 mg and 3 mg: 1 and 3 mg PGE2/kg/d daily sc injection. Ec-%Er.Pm: percent endocortical eroded perimeter, Ec-%L.Pm: percent endocortical mineralized perimeter, Ec-MAR: endocortical mineral apposition rate, Ec-BFR: bone formation rate per unit of endocortical surface. Ec-%L.Pm/%Er.Pm: ratio of percent mineralized perimeter to percent eroded perimeter of endocortical surface; Ec-BFR/%Er.Pm: ratio of bone surface bone formation rate to percent eroded perimeter of endocortical surface.

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porosity exceeded the bone gain from stimulated periosteal bone formation. Alternatively, the influence of intermittent PGE2 for 21 days on cancellous bone was mostly anabolic. Bone turnover and remodeling were stimulated along with positive cancellous bone balance and a shorter resorption period (the magnitude of the formation phase is greater than that of the resorption phase). In the cortical bone, the intermittent treatment did not increase bone mass because of insufficient treatment time to generate measurable bone gain. However, it stimulated positive endocortical bone balance-induced bone gain that was offset by increased intracortical porosity and decreased periosteal apposition. Both routes of administration increased bone turnover (BFR/ BV) and remodeling (activation frequency). Continuous PGE2 administration was more efficacious than intermittent administration with a continuous to intermittent ratio for bone turnover of 1.3 and activation frequency of 2.6 (Table 2). This suggests more BMUs were available to be subjected to the negative endosteal bone balance to generate the reduced cancellous bone mass in 2 remodeling cycles. The lower relative activation frequency with intermittent treatment exposed BMUs to positive endosteal bone balance only was able to generate a non-significant 9% increase with 1 mg and a significant 11% increase with the 3 mg dose. Factors contributing to the negative cancellous bone balance induced by continuous PGE2 administration partially involved the imbalance caused by stimulation of bone resorption (+161%) greater than bone formation (+31%) coupled with a shorter formation period (14%). The former was possibly due to the imbalance in RANKL to osteoprotegerin ratios in favor of RANKL [17] and the latter was due to increased osteoblastic apoptosis [18]. In contrast, the intermittent PGE2-induced positive cancellous bone balance was partly due to stimulated cancellous bone formation (+65%) coupled with a shorter resorption period (36%). The latter response may be due to accelerated osteoclast apoptosis [18]. Factors contributing to the cortical bone loss induced by continuous PGE2 was attributed mainly to stimulated endocortical bone resorption causing a negative bone balance that offset the stimulated bone formation the magnitude of endocortical bone resorption (+ 321%) was greater than the periosteal bone formation (+ 132%). In contrast, the lack of effect of intermittent PGE2 was due to the depression of periosteal apposition and increased intracortical bone porosity that offset the stimulated endocortical bone formation (+ 200%), with its positive bone balance of + 105% and + 176%. Another effect of continuous PGE2 administration was the robust stimulation of periosteal bone formation opposed to the decrease with intermittent treatment. The latter was a surprise since previous studies have shown intermittent PGE2 markedly increased periosteal bone formation in the tibial shaft of male, intact and ovariectomized female rats [2,3,6,1925]. One possible explanation is that this is a site-specific response of lumbar vertebral cortices since this is the only report to date for this site. More studies are needed to confirm this. Regardless, the powerful ability of continuous PGE2 to stimulate periosteal bone formation should improve bone strength. It is generally

accepted that periosteal expansion is an effective mechanism to dramatically improve vertebral bone strength and reduce fracture risk [2630]. Both continuous and intermittent PGE2 stimulated intracortical bone remodeling to increase intracortical porosity. The continuous route was found to be 1.5 times more efficacious than by intermittent PGE2 (Table 3). The intracortical porosity activated by both routes of administration was localized near the endocortical surface adjacent to the marrow [19,3133] with little influence on bone strength. Pores known to be close to endocortical surfaces have less effect on mechanical properties than pores near the periosteal surfaces [34]. In addition, these were transiently increased intracortical pores refilled on cessation of treatment [21,35]. In our previous paper on the effects of continuous versus intermittent PGE2 treatment on the proximal tibial metaphysis and tibial shaft, we proposed a treatment pattern in which continuous PGE2 treatment could become anabolic by cotreating with an anti-catabolic agent to suppress continuous PGE2-induced bone resorption [25]. Briefly, the plan emphasizes the beneficial effect of periosteal expansion and conversion of negative to positive endosteal bone balance (the magnitude of bone formation phase greater than that of resorption phase), an approach to add cortical bone to the lumbar vertebral body, which intermittent PGE2 did not. In this scheme, we assumed that combining continuous PGE2 with an anti-catabolic agent would have a better effect than intermittent PGE2. The effects of co-treatment with intermittent PGE2 and an anti-catabolic agent have been shown to be equal to or better than the intermittent PGE2 alone [2224,36,37]. Thus, we postulated that cotreatment of continuous PGE2 and an anti-catabolic agent would continue to increase periosteal modeling, refill endosteal remodeling spaces and intracortical porosity, and initiate positive endosteal bone balance. This would lead to an increase in cancellous and cortical bone mass, architecture and bone geometry to yield increased bone strength. At cessation of treatment, there would be refilling of intracortical porosity and remodeling spaces with maintenance of the increased periosteal and cancellous bone mass furthering increase of bone strength [21,35]. Whether this strategy employing continuous PTH to the clinical relevant site, the lumbar vertebra, would increase bone strength is worth pursuing. There are two obvious limitations to the current study. One is the lack of pre-treatment baseline groups. The animals labeled as baseline controls were sacrificed at 9 days into the study. Without a true baseline group, we avoided comparing the terminal changes with the baseline group to avoid making any misinterpretation. Secondly, we did not attempt to quantify the PGE2 activation of modeling and remodeling. Previously we reported that much of the intermittent PGE2-induced bone formation was driven more by modeling than by remodeling bone gain [7,8,37]. Thus, it would be informative in the current study if we were able to determine to what degree continuous PGE2 differs from intermittent PGE2 in influencing modeling and remodeling bone formation. In summary, continuous PGE2 exposure led to bone catabolism while intermittent administration led to bone anabolism.

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Both routes of administration stimulated bone turnover and remodeling to expose more BMUs to either negative or positive endosteal bone balance. The net catabolic effect of continuous administration was the result of the imbalance in bone gain and loss in favor of bone loss. The magnitude of the increased periosteal bone formation-induced bone gain was less than the magnitude of the increased negative endosteal bone balance and intracortical porosity-induced bone loss. The anabolic effect of intermittent administration was the result of imbalance in bone gain over loss. The magnitude of the increased positive endosteal bone balance-induced gain was more than the magnitude of the decreased periosteal bone expansion and increased intracortical porosity. The principal effects of continuous administration were the robust stimulation of bone resorption and periosteal bone formation while that for intermittent administration was the stimulation of endosteal bone formation. In addition, this is the first study to show the lack of intermittent PGE2 stimulation of periosteal apposition in lumbar vertebral bodies, while intermittent PGE2 shortened the resorption period and continuous PGE2 shortened the formation period. Lastly, a plan to convert continuous PGE2 into an anabolic agent with co-treatment with an anti-catabolic agent to generate lumbar vertebral cortical bone with increased bone strength was proposed, a much needed approach to increase vertebral cortical bone mass and strength. References
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